Neisseria gonorrhoeae causes a very common sexually transmitted disease in men and women that often results in severe consequences for women including pelvic inflammatory disease, ectopic pregnancy, and infertility. The gonococcal population is becoming increasingly antibiotic resistant, and multidrug resistant strains have arisen. This proposal seeks to understand the mechanisms of peptidoglycan breakdown involved in cell separation and to develop cell separation as a target for antimicrobial therapy. Preliminary data indicate that defects in cell separation make N. gonorrhoeae more sensitive to antibiotics as well as defective in infection of human cells. Previously we demonstrated that mutations in genes for peptidoglycan degradation - amiC or ltgC - in N. gonorrhoeae cause aberrant growth, loss of cell separation, and increased autolysis. These mutants grow in large aggregates sharing a single interconnected cell wall and with a single outer membrane surrounding the aggregate. Here we will 1) determine the biochemical functions of two peptidoglycan-degrading enzymes, AmiC and LtgC, required for cell separation and develop assays that could be used to identify inhibitors of these enzymes, 2) examine the localization and possible interactions of AmiC and LtgC in gonococcal cells in order to determine the function of these enzymes for cell separation and develop assays for these enzymes in cells, and 3) determine the extent of defects of cell separation mutants for infection of human cells and for resistance to antibiotics. Knowledge gained in this study and assays developed here will facilitate the identification of antibiotics for the treatment of gonococcal diseases and for combating other bacteria that have similar mechanisms of cell separation.